Extracellular ATP acts as a neurotransmitter and modulator of cellular function by binding to and activating two classes of cell surface receptors: P2X ion channels and P2Y G protein-coupled receptors. The signaling function of ATP, ADP, UTP, UDP, UDP-sugars and other nucleotides by activating P2Y receptors is fertile ground for drug discovery, because of the important influence of these receptors on cell proliferation and death, cytokines and immune responses, stem cell differentiation, communication at the tripartite synapse in the nervous system, and many other functions. This class of receptors is responsible for maintaining homeostasis in a variety of organ systems, and their pharmacological manipulation is especially relevant to chronic disease states such as inflammation, digestive disorders, neurodegeneration, endocrine disorders and cardiac failure. The challenge to the medicinal chemist is both to enhance selectivity of the nucleotide derivatives within the family of eight P2Y receptor subtypes and to enhance the stability and bioavailability of normally unstable native ligands. The introduction of numerous selective agonists and antagonists of P2Y receptors (P2YRs) by our laboratory, and their availability for use as pharmacological research tools, has greatly facilitated research in this area. Exploration of the role of P2YRs in large part using ligand designed and synthesized in our laboratory (given the number designation MRS, after the Molecular Recognition Section), which are mostly made available by us or by commercial suppliers to many other research laboratories. This availability has led to the introduction of new therapeutic concepts, such as use of P2Y1R antagonists as antithrombotic agents, P2Y2 agonists for cardioprotection, P2Y6R agonists or P2Y13R antagonist for treatment of diabetes, P2Y6R agonists for skeletal muscle protection and P2Y14R antagonists for asthma. For example, we have explored in detail the signaling pathways related to the action of UDP on the P2Y6R of pancreatic beta islet cells, including preventing apoptosis and promoting insulin release. Many of the known ligands for the P2YRs are notoriously unstable when administered in vivo due to the labile phosphate moiety. In order to explore new drug concepts for the P2YR, it is necessary to improve upon the bioavailability and stability of the compounds either by nucleotide modification or by searching for chemically diverse, preferably uncharged, ligands. Both of these efforts are being carried out in our laboratory using molecular modeling and organic chemical synthesis. Virtual screening has identified compound leads for novel antagonists of the P2Y1R and other subtypes. We have also achieved the structure-functional analysis of various P2YRs, by indirect means, using mutagenesis and molecular modeling based on other GPCR templates, while we await a crystallographic structural determination within this class of GPCRs. A uracil nucleoside phosphonate MRS2802 is being explored as a P2Y2R allosteric agonist. We also designed and synthesized new agonists of the P2Y6 nucleotide receptor using classical and computational approaches. The most potent of these agonists, UDP analogue MRS2795, contains a methanocarba ring system in place of the ribose moiety, which was prepared as a pure stereoisomer using a novel synthetic route. This ring system is locked in the South (S) envelope conformation, which is greatly preferred over other conformations in agonist recognition at the P2Y6R. Thus, the conformational requirements of the ribose moiety of UDP analogues in binding to the P2Y6R are very different from those of the P2Y1R, which prefers a North (N) conformation. 5-AMP-activated protein kinase (AMPK) and its pharmacological modulators have been targeted for treating type 2 diabetes. Extracellular uridine 50-diphosphate (UDP) activates P2Y6Rs in pancreatic beta-islet cells to release insulin and reduce apoptosis, which would benefit diabetes. We studied the role of P2Y6R in activation of AMPK in MIN6 mouse pancreatic beta-cells and insulin secretion. Treatment with a potent P2Y6R dinucleotide agonist MRS2957 (500 nM) activated AMPK, which was blocked by P2Y6R-selective antagonist. Also, MRS2957 induced phosphorylation of acetylcoenzyme A carboxylase (ACC), a marker of AMPK activity. A calcium chelator, calmodulin-dependent protein kinase kinase inhibitor and IP3 receptor antagonist attenuated P2Y6R-mediated AMPK phosphorylation revealing involvement of intracellular Ca2+ pathways. P2Y6R agonist induced insulin secretion at high glucose, which was reduced by AMPK siRNA. Thus, P2Y6R has a crucial role in beta-cell function, suggesting its potential as a therapeutic target in diabetes. 4-Alkyloxyimino derivatives of pyrimidine nucleotides display high potency as agonists of certain P2YRs. For example, we designed and synthesized the first selective agonist of the P2Y4R MRS4062, a N4-oxoimino derivative of CTP, and defined a new hydrophobic binding region of the P2Y4R. This modification of the uracil ring also is effective for the P2Y6R. In an effort to functionalize a P2Y6R agonist for fluorescent labeling, we probed two positions with various functional groups, including alkynes for click chemistry. Functionalization of extended imino substituents at the 4 position of the pyrimidine nucleobase of CDP preserved P2Y6R potency. Fluorescent Alexa Fluor 488 conjugate MRS4129 activated the human P2Y6R and was internalized with an EC50 of 9 nM, and exhibited high selectivity for this receptor over other uridine nucleotide-activated P2Y receptors. Flow cytometry detected specific labeling with 16 to P2Y6R-expressing but not to wild-type 1321N1 cells. Theoretical docking of MRS4129 to a homology model of the P2Y6R predicted electrostatic interactions between the fluorophore and extracellular portion of TM3. Thus, we have identified the N4-benzyloxy group as a structurally permissive site for synthesis of functionalized congeners leading to high affinity molecular probes for studying the P2Y6R. The P2Y14R is highly expressed in hematopoietic cells. Although the physiologic functions of this receptor remain undefined, it has been strongly implicated recently in immune and inflammatory responses. Lack of availability of receptor-selective high-affinity antagonists has impeded progress in studies of this and most of the eight nucleotide-activated P2Y receptors. We carried out the first systematic SAR studies on the P2Y14R leading to UDP analogues of nanomolar affinity, such as selective agonist MRS2905. A series of molecules recently were identified as antagonists at the P2Y14R. We re-synthesized one of these molecules, a 4,7-disubstituted 2-naphthoic acid derivative (PPTN), and studied its pharmacological properties in detail. The concentration-effect curve of UDP-glucose for promoting inhibition of adenylyl cyclase by the P2Y14R was shifted to the right in a concentration-dependent manner by PPTN with a KB of 434 pM. In contrast, 1 microM PPTN exhibited no agonist or antagonist effect at any other P2YR. PPTN blocked chemotaxis of differentiated HL-60 human promyelocytic leukemia cells promoted by UDP-glucose but not fMetLeuPhe. P2Y14Rpromoted chemotaxis of freshly isolated human neutrophils also was blocked by PPTN. Thus, PPTN is a highly selective high-affinity antagonist that is useful for interrogating the action of P2Y14R in physiologic systems. In summary, we emphasize four aspects of studying the P2Y receptors: 1) design and synthesis of novel and selective agonists and antagonists based on SAR; 2) protein structure-function studies; 3) exploring the novel biological role of such receptors; and 4) conceptualization of future therapeutics. In this effort, there is a tight coupling of organic synthetic methodology, structural biology, and pharmacology.